Clusterin antisense therapy for treatment of cancer

ABSTRACT

A method for providing antisense therapy which reduces the expression of clusterin to provide therapeutic benefits in the treatment of cancer comprising administering from 40 to 640 mg anti-clusterin antisense oligonucleotide to a patient in need of treatment for a cancer expressing clusterin is provided. The method may include administering chemotherapeutic agent or agents, radiotherapy, and/or hormone ablation therapy. The invention also encompasses pharmaceutical compositions formulated to provide a dosage of 40 to 640 mg, and use of antisense in formulating a medicament.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is related to US Patent Publications 20020128220 and20030166591 and PCT Publication WO 00/049937, all of which areincorporated herein by reference. This application claims priority fromU.S. Provisional Application No. 60/559,324 filed Apr. 2, 2004, and U.S.Provisional Application No. 60/649,326 filed Feb. 2, 2005, which areincorporated herein by reference.

FIELD OF INVENTION

This invention relates to the field of treating cancer, morespecifically to the treatment of cancer using an amount of antisenseoligodeoxynucleotide effective to result in tumour-appropriatebiodistribution and to result in tumour-appropriate biodistribution andto reduce the effective amount of clusterin in cancer cells with limitedside effects.

BACKGROUND OF THE INVENTION

Standard treatments for cancer vary depending on type of cancer, stageand location. For local disease, excision and/or radiation may be used.For systemic disease, chemotherapy may be used, and forhormone-dependent tumours, hormone ablation therapy is an option. Withthe exception of surgery, treatment success is limited by toxicity andresistance.

Research during the past decade has identified several proteins that maypromote progression and resistance by inhibiting apoptosis. Of specialrelevance to development of AI (androgen-independent) progression andhormone refractory prostate cancer (HRPC) are those survival proteinsthat are up-regulated after apoptotic triggers, such as androgenablation, that function to inhibit cell death.

Clusterin is a ubiquitous protein with a diverse range of proposedactivities. In prostate epithelial cells, expression of clusterinincreases immediately following castration, reaching peak levels in ratprostate cells at 3 to 4 days post castration, coincident with the onsetof massive cell death. These results have led some researchers to theconclusion that clusterin is a marker for cell death, and a promoter ofapoptosis. On the other hand, it has been observed that Sertoli cellsand some epithelial cells express high levels of clusterin withoutincreased levels of cell death. In Sensibar et al., Cancer Research 55:2431-2437, 1995, the authors reported on LNCaP cells transfected with agene encoding clusterin, and watched to see if expression of thisprotein altered the effects of tumor necrosis factor α (TNFα), to whichLNCaP cells are very sensitive. Treatment of the transfected LNCaP cellswith TNFα was shown to result in a transient increase in clusterinlevels for a period of a few hours, but these levels had dissipated bythe time DNA fragmentation preceding cell death was observed.

Clusterin is expressed in a number of tumour types including breast(Redondo, M.; Villar, E.; Torres-Munoz, J., et al. Am J Pathol (2000)157(2):393-9), non-small cell lung carcinoma (July, L. V.; Beraldi, E.;So, A. et al. Mol Cancer Ther (2004) 3(3):223-32.), prostate (Miyake,H.; Nelson, C.: Rennie, P. S.; et al. Cancer Res. (2000) 60(1):170-6),ovarian (Hough, C. D.; Cho, K. R.; Zonderman A. B. et al. Cancer Res.(2001) 61(10):3869-76), renal, and bladder (Miyake, H.; Gleave, M.;Kamidono, S.; Urology (2002) 59(1): 150-4).

Transfection studies in vivo result in the development of a phenotypethat is resistant to standard therapies. Inhibition studies result indelay in progression and the promotion of apoptosis, as well as inincreased chemotherapy and radiation therapy sensitivity.

Antisense treatment of cancer is still a relatively new science. Certainaspects such as optimal dosing and route of administration are still amatter of study (Gewirtz, A. M. Curr Opin Mol Ther. (1999) 1(3):297-306;and Dean, N. M.; Bennett, C. F.; Oncogene (2003) 22(56):9087-96).Continuous infusion is a requirement of the first generation ofantisense therapeutics, which are characterized by rapid clearance fromplasma and do not show significant pooling in target tissue. Theantisense therapeutics thus administered may not accumulate in targettissues sufficiently to down-regulate the genetic and/protein targets,particularly in solid tumours.

There is a need for therapies that have better tissue pharmacokineticsso that less frequent and inconvenient administration is required. Also,efficient delivery will reduce the risk of unwanted side effects andpotential toxicities. Effective systemic therapies must therefore haveexcellent biodistribution to tumour sites, lymph nodes, and other commonsites of metastases, to afford optimal treatment.

PCT Publication WO 00/049937 and PCT Publication WO 03/072591, which areincorporated herein by reference, describe the use of antisense therapywhich reduces the expression of clusterin in certain cancers.

SUMMARY OF THE INVENTION

In accordance with the present invention, it has now been determinedthat effective antisense therapy which achieves biodistribution totumour tissue and tissues involved in metastasis (lymph nodes) andreduces the expression of clusterin with no increase in toxicity isachieved by administration of 40 to 640 mg, more preferably 300 to 640mg clusterin antisense oligonucleotide to a patient in need of treatmentfor a cancer expressing clusterin. In particular, such antisense therapycan be applied in treatment of prostate cancer, lung cancer, renalcancer, bladder cancer, breast cancer, and cancers metastasizing throughlymph nodes.

In accordance with one aspect of the invention, there is provided amethod for treating cancer using clusterin antisense.

In accordance with another aspect of the invention, there is provided amethod for providing antisense therapy which reduces the expression ofclusterin to provide therapeutic benefits in the treatment of cancerincluding administering from 40 to 640 mg anti-clusterin antisenseoligonucleotide to a patient in need of treatment for a cancerexpressing clusterin.

The amount of anti-clusterin antisense oligonucleotide administered ispreferably from 300 to 640 mg.

The anti-clusterin antisense oligonucleotide may have a sequenceselected from among Seq. Id. Nos. 1-14, for exampleCAGCAGCAGAGTCTTCATCAT (SEQ ID No.: 1); ATTGTCTGAGACCGTCTGGTC (Seq. IDNo.: 2); or GCTGGGCGGAGTTGGGGGCCT (Seq. ID No.: 3). The amount ofanti-clusterin antisense oligonucleotide administered may be from 40 to640 mg, or 300-640 mg. Administration of the anti-clusterin antisenseoligonucleotide may be once in a seven day period, 3 times a week, ormore specifically on days 1, 3 and 5, or 3, 5 and 7 of a seven dayperiod of a treatment cycle.

The cancer being treated may be prostate cancer, bladder cancer, ovariancancer, lung cancer, renal cancer, melanoma, and pancreatic cancer. Thepatient being treated may suffer from metastases or lymph nodeinvolvement. The lung cancer may be non-small cell lung cancer (NSCLC).

The method may further include the administration of a chemotherapeuticagent or agents, and the anti-clusterin antisense oligodeoxynucleotidemay be administered before, after or during the chemotherapeutic agentor agents. The chemotherapeutic agents may be cisplatin and gemcitabine,taxotere, and/or paclitaxel.

The method may further include the administration of radiation therapy,instead of or along with chemotherapy. The anti-clusterin antisenseoligonucleotide may be administered before, after or during theadministration of radiation therapy.

The method may further include the administration of hormone ablationtherapy, and the anti-clusterin antisense oligonucleotide may beadministered before, after or during the hormone ablation therapy.

The method may include the anti-clusterin antisense oligonucleotidebeing administered in combination with hormone ablation therapy andchemotherapy.

According to another aspect of the invention, there is provided anantisense-containing pharmaceutical composition packaged in dosage unitform, said pharmaceutical composition including anti-clusterin antisenseoligonucleotide, wherein the amount of anti-clusterin antisenseoligonucleotide in each dosage unit is from 40 to 640 mg, or 300 to 640mg. The antisense-containing pharmaceutical composition may have asequence selected from among Seq. ID Nos. 1-12, and may be an injectablesolution or suspension, which may further contain sodium ions.

According to another aspect of the invention, there is provided the useof an anti-clusterin antisense in the manufacture of a medicament forthe treatment of cancer, wherein the medicament is formulated to delivera dosage of 40 to 640 mg, or 300-640 mg, of the antisense to a patient.The antisense may have a sequence selected from among Seq. ID Nos. 1-12.

The medicament may contain sodium ions, and/or be in the form of aninjectable solution.

The methods and pharmaceutical compositions of the inventionsurprisingly result in a lower than expected concentration of clusterinantisense provided to a human to achieve target tissue concentrations.Further, the methods and compositions of the invention result insurprisingly high reduction of clusterin mRNA and clusterin protein incancer tissue in cancer patients relative to untreated patients. Inparticular, such extensive down-regulation was unexpected given therequirement to penetrate the blood/prostate barrier.

In accordance with another aspect of the invention, there is providedthe use of clusterin antisense for the manufacture of a medicament forthe treatment of cancer.

Other aspects and features of the present invention will become apparentto those ordinarily skilled in the art upon review of the followingdescription of specific embodiments of the invention in conjunction withthe accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

In drawings which illustrate embodiments of the invention:

FIG. 1 shows concentrations of anti-clusterin antisense (Seq. ID. No 1)in human prostate tissue (Tissue PK) graphically represented for dosesof 40 mg, 80 mg, 160 mg, 320 mg and 480 mg;

FIG. 2 is a graphical representation of dose dependent decreases inprostate tumour clusterin expression measured by immunohistochemistry(IHC) and in situ hybridization;

FIG. 3 is a graphical representation showing IHC and computer scoring(Image Pro-Plus™) of all doses as compared to <2 month neoadjuvanthormone therapy and patients receiving no treatment;

FIG. 4 is a graphical representation showing the clusterin stainingintensity of untreated prostate cancer patient cells;

FIG. 5 is a graphical representation showing dose dependent decreases inprostate tumour clusterin mRNA expression as observed by in situhybridization (ISH), as compared to mRNA expression in patients having<2 month neoadjuvant hormone therapy;

FIG. 6 is a graphical representation showing clusterin stainingintensity of untreated prostate cancer patient cells;

FIG. 7 is a graphical representation showing suppression of clusterinprotein expression in lymph nodes; and

FIG. 8 is a graphical representation of the plasma pharmacokineticsresulting from a single 2 hour intravenous administration.

DETAILED DESCRIPTION OF THE INVENTION

Numerical values in the specification and claims of this applicationshould be understood to include numerical values which are the same whenreduced to the same number of significant figures and numerical valueswhich differ from the stated value by less than the experimental errorof the measurement technique used in the present application todetermine the value.

As used in the specification and claims of this application, the term“clusterin” refers to the glycoprotein originally derived from rattestes, and to homologous proteins derived from other mammalian species,including humans, whether denominated as clusterin or an alternativename. The sequences of numerous clusterin species are known. Forexample, the sequence of human clusterin is described by Wong et al.,Eur. J. Biochem. 221 (3), 917-925 (1994), and in NCBI sequence accessionnumber NM_(—)001831. In this sequence, the coding sequence spans bases48 to 1397.

Systematic administration of antisense clusterin oligodeoxynucleotide(ODN or ASO) in animals (including humans) bearing human prostatecancer, lung cancer such as non-small cell lung cancer (NSCLC), bladdercancer, melanoma, breast cancer, renal cancer, ovarian cancer, etc. iseffective for inducing apoptosis and delaying disease progression.Systemic administration of antisense clusterin ODN is also effective indelaying progression to androgen independence. Thus, an individualsuffering from a clusterin expressing tumour can be treated withchemotherapy, radiation therapy, and in case of hormone-regulatedtumours, by initiating androgen-withdrawal to induce apoptotic celldeath of tumour cells in the individual, and administering to theindividual a composition effective to inhibit expression of clusterin bythe tumor cells, thereby promoting apoptosis and/or delaying theprogression of tumor cells. Furthermore, combined use of antisenseclusterin plus cytotoxic or other chemotherapy (e.g. taxanes)synergistically enhances chemosensitivity in hormone refractory prostatecancer, lung cancers such as NSCLC, breast, ovarian, renal, etc.

Thus, one embodiment of the invention may further include administrationof chemotherapy agents or other agents useful in cancer therapy priorto, concurrent with, or subsequent to and/or additional antisense ODNsdirected at different targets in combination. For example, the antisensemay be administered in conjunction with radiotherapy, surgery, hormonetherapy, or chemotherapy.

Further administration may be of hormone ablating agents, or hormoneblocking agents. Such agents may be administered in combination with achemotherapeutic agent to achieve treatment goals.

A second antisense ODN which inhibits expression of an anti-apoptoticprotein other than clusterin may also be administered along with theantisense clusterin ODN. For example, antisense clusterin ODN may beused in combination antisense inhibitors to bcl family members or XIAPfamily members.

An antisense clusterin ODN can also be combined with more conventionalchemotherapy agents such as taxanes (Paclitaxel™ or Docetaxel™),mitoxanthrone, gemcitabine, cyclophosphamide, decarbazine, topoisomeraseinhibitors, platinum-based chemotherapies such as cisplatin,mitoxanthrone, angiogenesis inhibitors, differentiation agents, signaltransduction inhibitors, and ancillary agents in general (such as, butnot limited to, Flutamide™).

More than one chemotherapeutic agent may be used in combination withantisense clusterin oligodeoxynucleotide, for examplecisplatin/gemcitabine, cisplatin/paclitaxel, cisplatin/docetaxel, orcarboplatin/paclitaxel combinations as described in Schiller et al;, N.Eng. J. Med. 346: 92-98 (2002), or a carboplatin/gemcitabinecombination.

“Combination” means either at the same time and frequency, or moreusually, at different times and frequencies, as the ODN, as part of asingle treatment plan. A chemotherapeutic agent will be administeredaccording to the best practice known in the art, specific to that agent.For example, in one aspect, a course of treatment was composed ofinjections of antisense clusterin oligodeoxynucleotide (for exampleOGX-011 as defined below) administered over 2 hours on Days 1, 3, 5, 8,15, 22, and 29; flutamide is administered for 4 weeks daily at 250 mgorally tid beginning on Day 1 to prevent tumour flare, and a singleinjection of beserelin acetate at 6.3 mg is administered subcutaneouslyon Day 1. In another aspect, clusterin antisense such as OGX-011 may begiven as a 2 hr intravenous (IV) infusion at fixed doses starting at 480mg weekly after 3 loading doses on days 3, 5, and 7. Cisplatin may begiven at 75 mg/m2 IV on day 1, and gemcitabine at 1250 mg/m2 IV on days1 and 8, for a maximum of 6 21-day cycles. In another schedule,clusterin antisense such as OGX-011 may be given by 2 hr IV infusion atfixed doses starting at 40 mg weekly after loading on days 1, 3, and 5.Taxotere may be given at a standard dose, namely IV 30 mg/m²/week (w)for 5 out of 6 weeks or 75 mg/m² every 3 weeks.

Chemotherapeutic agents may therefore be used in combination with theantisense clusterin oligodeoxy nucleotide (ODN) according to an aspectof the invention, but yet be administered at different times, differentdosages, and at a different frequency, than the ODN is administered.

In other embodiments of the invention, a therapeutic antibody targetedto the particular cancer being treated may be used with theanti-clusterin antisense. One example is for example trastuzumab forbreast cancer.

It has also been found that antisense clusterin has beneficial effectsfor other cancer types. Specifically, antisense clusterin ODN enhanceschemosensitivity in human renal cell cancer, a normally chemoresistantdisease with no active chemotherapeutic agent having an objectiveresponse rate higher than 10%. Radiation sensitivity is also enhancedwhen cells expressing clusterin are treated with antisense clusterinODN. Thus, the antisense clusterin ODNs can be used to treat a varietyof cancer types in which expression of clusterin has been observed.

Antisense oligonucleotides useful in the present invention are speciesof oligonucleotide that interact with mRNA encoding clusterin in such away as to reduce the effective amount of clusterin in the cell. As usedin this application, the “effective amount of clusterin” is the amountof clusterin which is present in a form which is functional to provideanti-apoptotic protection. The effective amount of clusterin is reducedby the application of an antisense oligonucleotide complementary to themRNA encoding clusterin, without regard for the specific mechanism bywhich the reduction is achieved. Specific antisense species that areuseful for this purpose include CAGCAGCAGAGTCTTCATCAT (Seq. ID No. 1),ATTGTCTGAGACCGTCTGGTC (Seq. ID No.: 2), and GCTGGGCGGAGTTGGGGGCCT (Seq.ID No.: 3). A preferred antisense oligonucleotide the 21meroligonucleotide CAGCAGCAGAGTCTTCATCAT (Seq. ID No.: 1) which is targetedto the translation initiation codon and next 6 codons of the humanclusterin sequence (Genbank accession no: NM_(—)001831). Mostpreferably, this antisense oligonucleotide is used in the form of a2′-MOE modified clusterin antisense oligonucleotide as described in U.S.patent application Ser. No. 10/080,794 filed Feb. 22, 2002. Thisoligonucleotide, which is referred to herein as OGX-011, has aphosphorothioate backbone throughout. The sugar moieties of nucleotides1-4 and 18-21 (the “wings”) bear 2′-O-methoxyethyl modifications and theremaining nucleotides (nucleotides 5-17; the “deoxy gap”) are2′-deoxynucleotides. Cytosines in the wings (i.e., nucleotides 1, 4 and19) are 5-methylcytosines. Other antisense oligonucleotides that may beused in the present invention are those listed in Nos. 4 to 12 below,and those described in U.S. Pat. No. 6,383,808, which is incorporatedherein by reference.gcacagcagg agaatcttca t 21  Seq. ID No. 4gcacagcagc aggatcttca t 21  Seq. ID No. 5tggagtcttt gcacgcctcg g 21  Seq. ID No. 6ccttcagctt tgtctctgat t 21  Seq. ID No. 7agcagggagt cgatgcggtc a 21  Seq. ID No. 8atcaagctgc ggacgatgcg g 21  Seq. ID NO. 9gcaggcagcc cgtggagttg t 21  Seq. ID No. 10ttcagctgct ccagcaagga g 21  Seq. ID No. 11aatttagggt tcttcctgga g 21  Seq. ID No. 12

Phase I pharmacokinetic and pharmacodynamic studies conducted with this2′-MOE modified oligonucleotide in patients with prostate cancer priorto radical prostatectomy show that the biodistribution anddown-regulated in vivo is achieved at lower concentrations thanpredicted from in vitro and in vivo tests, allowing broader range anddecreased frequency of dosing of the therapeutic agent. Thus, inaccordance with the invention, there is provided a method for providingantisense therapy which reduces the expression of cluster in thetreatment of cancer including the step of administering from 40 to 640mg clusterin antisense oligonucleotide to a patient in need of treatmentfor a cancer expressing Clusterin, more preferably from 300 to 640 mg.Dosages are calculated typically by patient weight, and therefore a doserange of about 1-20 mg/kg, or about 2-10 mg/kg, or about 3-7 mg/kg, orabout 3-4 mg/kg could be used. This dosage is repeated at intervals asneeded, clinical concept is dosing once per week with 3 loading dosesduring week one of cycle one only. The amount of antisense ODNadministered is one that has been demonstrated to be effective in humanpatients to inhibit the expression of clusterin in cancer cells.

The invention further provides antisense containing pharmaceuticalcompositions packaged in dosage unit form, wherein the amount in eachdosage unit is from 40 to 640 mg of the clusterin antisenseoligonucleotide, more preferably from 300 to 640 mg. Dosage units maybe, without limitation, injectable solutions or suspensions includingsolutions in bags for IV administration through an existing port,catheter or IV site, and powders or concentrates for preparation ofinjectable solutions. A formulation of particular usefulness is oneincluding sodium salts in an aqueous solution, for use in intravenous,subcutaneous, intraperitoneal, or intratumour administration.

The invention will now be further described with reference to thefollowing, non-limiting examples.

Example 1 Enhancing Hormone Sensitivity Using Various Clusterin ASOs inMurine Model

Targeting cell survival genes up-regulated by androgen withdrawal mayenhance castration-induced apoptosis and thereby prolong time to overtrecurrence. ASOs corresponding to the clusterin translation initiationsite were designed and shown to reduce clusterin levels in adose-dependent and sequence-specific manner (Miyake, H.; Nelson, C.;Rennie, P. S. et al. Cancer Res. (2000) 60(1):170-6; Miyake, H.; Chi,K.; Gleave, M. E. Clinical Cancer Res (2000) 6:1655-63.

Male mice bearing Shionogi tumours were castrated 2 to 3 weeks aftertumour implantation, at which time tumours were 1 to 2 cm in diameter,and randomly selected for treatment with antisense clusterin versusmismatch control oligonucleotides. Beginning 1 day after castration,12.5 mg/kg of oligonucleotide was administered once daily byintraperitoneal injection for 40 days. Shionogi tumours regressed fasterand complete regression occurred earlier in mice treated with clusterinASO compared to controls. Furthermore, clusterin ASO treatmentsignificantly delayed recurrence of AI tumours compared to mismatchcontrol oligonucleotide treatment. After an observation period of 50days post-castration, mean tumour volume in the mismatch-treated controlgroup was 6-times that of the ASO-treated group. Dose-dependent studiesusing mouse clusterin ASOs in the murine Shionogi model demonstrateddose-dependent activity up to 25 mg/kg/day, but did not reveal anyobvious toxicity in mice treated with up to 50 mg/kg/day. Analysis oftumours excised 3 days after castration confirmed that ASO treatedtumours had lower clusterin levels and earlier evidence of PARP cleavagefragments, consistent with earlier onset of castration-inducedapoptosis. Changes in clusterin mRNA levels in various normal mouseorgans after ASO treatment were also evaluated. Shionogi tumours,spleen, kidney, brain and prostate were harvested 3 days post-castrationfor RNA extraction from mouse administered ASO or mismatch control underthe same treatment schedule described above.

Murine clusterin ASO had no effect on clusterin expression levels inspleen, kidney and brain, whereas clusterin mRNA expression in tumourand prostate tissues was significantly lower after ASO administrationcompared to mismatch control (Miyake H (a), 2000).

Example 2 Characterization of Human Clusterin ASO-OGX-011

ASO sequences directed against different sites on the human clusteringene were synthesized and tested for their ability to decrease clusteringene expression in human prostate cancer cell lines that overexpressclusterin (PC3 and LNCaP). The clusterin gene was ‘walked’ using aquantitative TaqMan RT-PCR assay, which compared the potency of over 80clusterin ASOs. An active and novel 21-mer ASO(5′-CAGCAGCAGAGTCTTCATCAT-3′; SEQ ID NO. 1), complementary to thetranslation initiation site of human clusterin, was capable ofinhibiting clusterin mRNA expression by up to 90%. OGX-011 has thisoptimal sequence, and is a 2nd-generation ASO using a phosphorothioatebackbone and an additional 2′-O-(2-methoxy) ethyl (2′-MOE) modificationto the 2′-position of the carbohydrate moiety on the 4 nucleotides atthe 5′ and 3′ ends of the molecule (gapmer).

Example 3 Enhancing Chemotherapy Sensitivity Using Clusterin ASOs inProstate Cancer

Clusterin ASOs also increased the cytotoxic effects of mitoxantrone andpaclitaxel, reducing the IC50 of PC3 and Shionogi cells by 75%-90%(Miyake, H.; Rennie, P.; Nelson C. et al. (2000) Cancer Res 60:2547-54;and Miyake, H.; Chi, K.; Gleave, M. E. (2000) Clinical Cancer Res6:1655-63). Cytotoxicity of 10-nM paclitaxel increased in adose-dependent manner as the concentration of clusterin ASO increased.The induction of apoptosis by 10-nM paclitaxel, as demonstrated by DNAladdering and PARP cleavage, could only be seen when used with clusterinASOs.

Synergy between clusterin ASOs and chemotherapy also occurs in vivo. Therecurrence of AI Shionogi tumours is delayed longest with combinedclusterin ASOs and paclitaxel compared to either agent alone. Althoughclusterin ASOs had no effect on the growth of established AI Shionogi orPC3 tumours, clusterin ASOs synergistically enhanced paclitaxel-inducedtumour regression in both the Shionogi and human PC3 models (Miyake, H.2000; Miyake, H, 2000, as above).

Example 4 Enhancing Chemotherapy Sensitivity Using Clusterin ASOs inRenal Cancer

Clusterin may also play a role in mediating chemoresistance in renalcell carcinoma and other tumours. ASO decreased clusterin levels inhuman renal cell cancer Caki-2 cells in a dose-dependent andsequence-specific manner. Pretreatment of Caki-2 cells with theseclusterin ASO's significantly enhanced chemosensitivity to paclitaxel invitro. Characteristic apoptotic DNA-laddering occurred after combinedtreatment with OGX-011 plus paclitaxel, but not with either agent alone.In vivo administration of OGX-011 enhanced in a synergistic fashionpaclitaxel-induced Caki-2 tumour regression and delayed tumourprogression by 50%.

Example 5 Enhancing Radiation-Sensitivity Using Human Clusterin ASOs InVitro

Clusterin-overexpressing LNCaP cells were less sensitive to irradiationwith significantly lower cell-death rates (23% after 8 Gy) compared toparental LNCaP cells (50% after 8 Gy) 3 days after irradiation(Zellweger, T.; Chi, K.; Miyake, H.; et al. Clin Cancer Res. (2002)8(10):3276-84). Clusterin expression in PC-3 cells after radiationincreased in a dose-dependent manner in vitro by 70% up to 12 Gy and invivo by >80%. Inhibition of clusterin expression in PC-3 cells usingclusterin ASO's before radiation significantly decreased PC-3 cellgrowth rate and plating efficiency, and enhanced radiation-inducedapoptosis. In vivo administration of clusterin ASO before and afterradiation significantly reduced PC-3 tumour volume by 50% at 9 weeks ascompared to mismatch control oligonucleotides.

These findings support the hypothesis that clusterin acts as a cellsurvival protein that mediates radioresistance through the inhibition ofapoptosis.

Example 6 OGX-011 Pre-Clinical Data

As a class, the second-generation phosphorothioate 2′-O-methoxyethylribose gapmer antisense oligonucleotides have pharmacokinetic andtoxicity profiles favorable for the intended clinical use.Pharmacokinetics of these compounds are characterized by rapid clearancefrom plasma following intravenous administration and broad distributionto tissues. Once distributed to tissues, these second generation ASO arecleared slowly. Tumour concentrations of an unmodified (firstgeneration) phosphorothioate clusterin ASO corresponding to SEQ ID NO. 4was compared to OGX-011. Using the PC3 model, animals were treated withASO, tumours excised 24, 72, 120 and 168 hours after last dosing andtumour ASO concentration assessed by capillary gel electrophoresis. At24 hours, OGX-011 concentration was ten times greater than that ofunmodified ASO (755+261 nM vs 60+10 nM). In addition, only 10% of theunmodified phosphorothioate ASO were full length while 90% of theOGX-011 ASO detected in the tumours were full length. At 120 hours afterthe last treatment, the phosphorothioate ASO were not detectable,however OGX-011 remained detectable at 120 hours (632+192 nM) and 168hours (180+14.1 nM) and also was predominantly full length(approximately 90% of detected ASO) (Zellweger, T.; Miyake, H.; Cooper,S.; et al. J Pharmacol Exp Ther (2001) 298:934-40).

In animal toxicology and pharmacokinetic studies, OGX-011 wasadministered by I.V. injection at doses of 1, 5, 20 and 50 mg/kg to CD-1mice, and 1-hour I.V. infusion at 1, 3, and 10 mg/kg to rhesus monkeys.In both species, the dosing period was 4 weeks with alternate day dosingfor the first 4 doses (a “loading period”) and weekly thereafter (a“maintenance period”). This dosing regimen was based on existingknowledge about the long tissue half-lives of this class of compound.

No clinical signs of toxicity were observed after 4 weeks of exposure atdoses up to 50 mg/kg in mice or 10 mg/kg in monkeys. Despite similarlevels of tissue exposure in liver achieved in monkeys, mice weregenerally more sensitive to liver toxicities as indicated by elevatedtransaminases. Many of the toxicities observed in mice were thought tobe related to a generalized mild immunostimulation, an effect ofphosphorothioate oligonucleotides that is generally more prominent inrodents than in primates (Parker S L. Cancer Statistics 65: 5-27, 1996).

Genetic toxicity studies (in vitro bacterial cell gene mutation andmouse lymphoma gene mutation) were negative.

Example 7 Changes in Clusterin Expression in Human Prostate CancerSpecimens after Neoadjuvant Hormone Therapy (NHT)

Materials:

Buserelin acetate (Aventis). Each implant dose, in the form of twocream-filled rods, contains a total of 6.6 mg buserelin acetate (6.3 mgbase) and 26.4 poly-(D,L-lactide-co-glycolide) in a 75:25 molar ratio.Both rods are implanted in one operation into the subcutaneous tissue ofthe lateral abdominal wall.

Flutamide is an approved commercially available non-steroid agent thatspecifically blocks androgen-binding receptors.

Methods and Results:

To define temporal changes in clusterin in human prostate cancer, 128radical prostatectomy specimens from patients after 0, 3 or 8 months ofNHT were stained for clusterin using immunohistochemistry (July L V,Akbari M, Zellweger T, et al. Prostate 50: 179-188, 2002). Residual fociof cancer cells from radical prostatectomy specimens treated with NHTexhibited strongly positive staining (intensity+3, +4) for clusterin in80-90% of the surviving cancer cells compared to either absent (0) orlow intensity staining (+1 to +2) in 10-20% of cancer cells in non-NHTtreated specimens. Consistent with previous reports, staining forclusterin was found in the cytoplasm of luminal epithelial cells, and nonuclear staining was observed.

Western blot analyses of clusterin were performed on prostate cancerspecimens from untreated patients (n=5), NHT treated patients (n=5) andtwo patients with androgen independent disease. To compare changes inprotein expression, a normalization procedure against the cytokeratin tovimentin ratio was performed because of increases in the stromal toepithelial ratio that occur with NHT. Normalized clusterin levels weresignificantly higher in all treated patients compared with untreatedpatients, increasing 17-fold after androgen ablation. These data confirmthat clusterin increases in prostate cancer cells after immediatelyfollowing androgen withdrawal and support the hypothesis that clusterinserves as a protective against the apoptotic stimulus of androgenablation.

Example 8 Safety, Plasma and Tissue Pharmacokinetics, and TissuePharmacodynamics of OGX-011 in Prostate Cancer Patients

Materials

Phosphorothioate 2′-O-methoxyethyl ribose ASO targeted to thetranslation initiation site of clusterin mRNA (designated OGX-011), isformulated as OGX-011 Injection, 20 mg/mL, in an isotonic,phosphate-buffered saline solution (pH 7.4) for intravenous (I.V.)administration; the drug product solution may be diluted into salineprior to I.V. administration. The formulation details are listed inTable 1.

Buserelin acetate (Aventis)—see above.

Flutamide—see above.

TABLE 1 Ingredients Concentration OGX-011 20 mg/mL Dibasic sodiumphosphate, heptahydrate, USP 14.33 mg/mL Monobasic sodium phosphate,monohydrate, USP 1.73 mg/mL Sodium chloride, USP 2.70 mg/mL HydrochloricAcid, NF pH adjustment Sodium Hydroxide, NF pH adjustment Water forInjection, USP q.s. to 1.0 mLMethods:

Patients having localized prostate cancer with high-risk features andcandidates for prostatectomy were enrolled to this dose escalationtrial. Twenty patients were treated and evaluated.

Each course of treatment was composed of injections of OGX-011administered over 2 hours on Days 1, 3, 5, 8, 15, 22, and 29 using fixeddose escalation plan starting at 40 mg; 4 weeks of daily flutamide 250mg orally tid beginning on Day 1 to prevent tumour flare, and a singleinjection of beserelin acetate 6.3 mg subcutaneously on Day 1. Eachpatient received a maximum of 1 course only.

Prostatectomy was performed day 30-36. Prostate tissue concentrations ofOGX-011 were determined by a fully validated ELISA method. Briefly,clusterin mRNA in human prostate tissues were determined by in situhybridization using standardized controls including OGX-011 and humanplasma (EDTA) (Biochemed), and reagents including Qiagen cutting Probe112989 cut (5′ end biotinated, 3′ end is labeled with digoxigenin), S1nuclease (Life Technologies), anti-digoxigenen-Fab fragments conjugatedwith alkaline phosphatase (AP), AttoPhos® fluorescent substrate andAttoPhos® reconstitution buffer, and Reacti-Bind™ NeutrAvidin™ (Pierce)coated Nalgene™ Nunc™ flat-bottomed polystyrene 96-well microwell platesblocked with SuperBlock®. Various label-recommended buffers and saltswere also used, purchased from various sources such as FisherScientific, Laboratoire MAT, Sigma, VWR, BDH, Pierce, and BioSource.Polypropylene containers were used to prepare and store all referencestandard solutions.

Clusterin protein in human prostate tissues was also determined by:

1. IHC using visual scoring of tumour cells conducted by two independentpathologists. Specimens were graded from 0 to +3 intensity, representingthe range from no staining to heavy staining and the overall percentageof cancer cells showing staining (0-100%) was indicated. The mean of thetwo pathologists' scores (the product of intensity and percentage ofcells) was used for reporting and standard error reported; and2. IHC using an automated scoring system of tumour cells conducted by anImage Pro-Plus™ system. The scoring system used by Image Pro™ can bedefined by the user to provide greater gradations and provide additionalindependent semi-quantitative data.Results:

Toxicity was limited to grade 1 or 2, and included fevers, rigors,fatigue and transient AST and ALT elevations at higher doses of OGX-011.Plasma pharmokinetics analysis showed linear increases in AUC with at1/2 of approximately 2 hours.

The 40 mg, 80 mg and 160 mg dose cohorts achieved mean concentrations ofOGX-011 that were approximately equivalent and thus not dose dependent.OGX-011 concentrations increased to a mean of 1.668 :g/g (˜227 nM) at320 mg and 2.30 :g/g (˜313 nM) at 480 mg dosing, representing a 4.9 foldincrease and 6.76 fold increase at the 320 mg and 480 mg dose,respectively.

As measured by immunohistochemistry, each of the 320 mg and 480 mg dosecohorts achieved a 30% decrease in clusterin protein expression ascompared to <2 month neoadjuvant hormone therapy. The mean percent ofdown-regulation of clusterin protein in the 320 mg and 480 mg dosecohorts was 31.2% and 70.6%, respectively. The mean percent age ofcancer cells in untreated prostate cancer patients staining intensity“0”, or no clusterin protein, was 14.25%, and cells staining intensity“3,” (high clusterin protein) was 18.75%. Following <2 months ofneoadjuvant hormone therapy, the percentage of cancer cells stainingintensity 0 decreased to 1.5%, and cells staining intensity 3 increasedto 48%. Surprisingly, the mean percentage of cancer cells stainingintensity 0, 1, 2 and 3 in prostate cancer patients receiving 320 mgOGX-011 was restored to approximately equivalent levels to untreatedcontrols. The mean percentage of cancer cells staining intensity 0increased dramatically in patients treated with 480 mg of OGX-011 to48.17%. In this same dose cohort, the percentage of cancer cellsstaining intensity 3 decreased dramatically to 6.56%.

IHC and computer scoring (Image Pro-Plus™) of all doses as compared to<2 month neoadjuvant hormone therapy showed a mean percent downregulation of clusterin protein in the 320 mg and 480 mg dose cohorts of68.7% and 76.8%, respectively.

Example 9 Antisense Oligonucleotide to Clusterin (OGX-011) inCombination with Gemcitabine and Cisplatin As First-Line Treatment ForPatients With Advanced Non-Small Cell Lung Cancer (NSCLC)

Patients with previously untreated stage IIIB or IV NSCLC were eligible.OGX-011 was given as a 2 hr IV infusion at fixed doses starting at 480mg weekly after 3 loading doses on days 7, 5, and 3). Cisplatin wasgiven at 75 mg/m2 IV on day 1, and gemcitabine at 1250 mg/m2 IV on days1 and 8. A maximum of 6 21-day cycles were performed.

Serum clusterin levels were measured at baseline and on day 1 of eachcycle. Where possible, pre- and post-treatment (cycle 2, day 1) biopsiesof the tumours were taken. The toxicities observed were typical forcisplatin/gemcitabine therapy alone.

The combination of OGX-011 and cisplatin/gemcitabine is more effectivethan either treatment alone.

The invention claimed is:
 1. A method for providing antisense therapywhich reduces the expression of clusterin to provide therapeutic benefitin the treatment of cancer comprising administering from 480 mg to 640mg anti-clusterin antisense oligonucleotide intravenously to a human inneed of treatment for a cancer expressing clusterin, which human alsoreceives hormone ablation therapy, radiation therapy or achemotherapeutic agent, wherein the nucleotide sequence of theanti-clusterin antisense oligonucleotide is CAGCAGCAGAGTCTTCATCAT (Seq.ID No.: 1), wherein the anti-clusterin antisense oligonucleotide has aphosphorothioate backbone throughout, has sugar moieties of nucleotides1-4 and 18-21 bearing 2′-O-methoxyethyl modifications, has nucleotides5-17 which are 2′ deoxynucleotides, and has 5-methylcytosines atnucleotides 1, 4, and
 19. 2. The method of claim 1, wherein the amountof anti-clusterin antisense oligonucleotide administered is 640 mg. 3.The method of claim 1, wherein the anti-clusterin antisenseoligonucleotide is administered once in a seven day period.
 4. Themethod of claim 1, wherein the anti-clusterin antisense oligonucleotideis administered on day 1, 3 and 5 of the first seven day period of atreatment cycle, and once in a seven day period thereafter.
 5. Themethod of claim 1, wherein the cancer is prostate cancer, bladdercancer, ovarian cancer, lung cancer, renal cancer, melanoma, orpancreatic cancer.
 6. The method of claim 5, wherein the human suffersfrom metastases.
 7. The method of claim 5, wherein the human suffersfrom cancer with lymph node involvement.
 8. The method of claim 1,wherein the human receives a chemotherapeutic agent.
 9. The method ofclaim 8, wherein the chemotherapeutic agent is gemcitabine.
 10. Themethod of claim 8, wherein the chemotherapeutic agent is carboplatin.11. The method of claim 8, wherein the chemotherapeutic agent ispaclitaxel.
 12. The method of claim 8, wherein the chemotherapeuticagent is paclitaxel and carboplatin.
 13. The method of claim 8, whereinthe chemotherapeutic agent is docetaxel.
 14. The method of claim 8,wherein the chemotherapeutic agent is prednisone.
 15. The method ofclaim 8, wherein the chemotherapeutic agent is mitoxantrone.
 16. Themethod of claim 1, wherein the chemotherapeutic agent is mitoxantroneand prednisone.
 17. The method of claim 1, wherein the chemotherapeuticagent is docetaxel and prednisone.
 18. The method of claim 1, whereinthe cancer is non-small cell lung cancer.
 19. The method of claim 1,wherein the cancer is prostate cancer.
 20. The method of claim 1,wherein the cancer is renal cancer.
 21. The method of claim 1, whereinthe cancer is breast cancer.
 22. The method of claim 12, wherein thecancer is non-small cell lung cancer.
 23. The method of claim 22,wherein the amount of anti-clusterin antisense oligonucleotideadministered is 640 mg.
 24. The method of claim 23, wherein theanti-clusterin antisense oligonucleotide is in an aqueous solutioncomprising sodium ions.
 25. The method of claim 17, wherein the canceris prostate cancer.
 26. The method of claim 25, wherein the amount ofanti-clusterin antisense oligonucleotide administered is 640 mg.
 27. Themethod of claim 26, wherein the anti-clusterin antisense oligonucleotideis in an aqueous solution comprising sodium ions.
 28. The method ofclaim 16, wherein the cancer is prostate cancer.
 29. The method of claim28, wherein the amount of anti-clusterin antisense oligonucleotideadministered is 640 mg.
 30. The method of claim 29, wherein theanti-clusterin antisense oligonucleotide is in an aqueous solutioncomprising sodium ions.
 31. A method for providing antisense therapywhich reduces the expression of clusterin to provide therapeutic benefitin the treatment of prostate cancer comprising administeringintravenously 640 mg anti-clusterin antisense oligonucleotide in anaqueous solution once in a seven day period to a human in need oftreatment for prostate cancer expressing clusterin, which human alsoreceives docetaxel and prednisone, wherein the nucleotide sequence ofthe anti-clusterin antisense oligonucleotide is CAGCAGCAGAGTCTTCATCAT(Seq. ID No.: 1), wherein the anti-clusterin antisense oligonucleotidehas a phosphorothioate backbone throughout, has sugar moieties ofnucleotides 1-4 and 18-21 bearing 2′-O-methoxyethyl modifications, hasnucleotides 5-17 which are 2′deoxynucleotides, and has 5-methylcytosinesat nucleotides 1, 4, and
 19. 32. A method for providing antisensetherapy which reduces the expression of clusterin to provide therapeuticbenefit in the treatment of non-small cell lung cancer comprisingadministering intravenously 640 mg anti-clusterin antisenseoligonucleotide in an aqueous solution once in a seven day period to ahuman in need of treatment for non-small cell lung cancer expressingclusterin, which human also receives paclitaxel and carboplatin, whereinthe nucleotide sequence of the anti-clusterin antisense oligonucleotideis CAGCAGCAGAGTCTTCATCAT (Seq. ID No.: 1), wherein the anti-clusterinantisense oligonucleotide has a phosphorothioate backbone throughout,has sugar moieties of nucleotides 1-4 and 18-21 bearing2′-O-methoxyethyl modifications, has nucleotides 5-17 which are2′deoxynucleotides, and has 5-methylcytosines at nucleotides 1, 4, and19.